Download Differential GH-releasing hormone regulation of GHRH receptor

Am J Physiol Endocrinol Metab
280: E626–E631, 2001.
Differential GH-releasing hormone regulation of
GHRH receptor mRNA expression in the rat pituitary
CATHERINE M. LASKO,1* ANDREW I. KORYTKO,1*
WILLIAM B. WEHRENBERG,3 AND LEONA CUTTLER1,2
Departments of 1Pediatrics and 2Pharmacology, Case Western Reserve University,
Cleveland, Ohio 44106; and 3College of Agriculture, Forestry, and Life Sciences,
Clemson University, Clemson, South Carolina 29634
Received 26 May 2000; accepted in final form 15 December 2000
THE GROWTH HORMONE-RELEASING HORMONE
(GHRH) receptor plays a critical role in somatotroph function by
* C. Lasko and A. Korytko contributed equally to this work.
Address for reprint requests and other correspondence: L. Cuttler,
Dept. of Pediatrics, Rainbow Babies and Children’s Hospital, Rm.
737, Case Western Reserve University, 11100 Euclid Ave., Cleveland, OH 44106-6004.
E626
mediating the stimulatory effects of GHRH on GH
synthesis and secretion (3, 14, 15). Although there are
several clear examples of hypothalamic secretagogues
influencing expression of their pituitary receptors (27,
36, 37), data on the GHRH/GHRH receptor axis have
been difficult to interpret due to apparent incongruities
in findings. For example, in perinatal rats, short-term
GHRH antiserum treatment of adult rats has been
reported to increase GHRH receptor mRNA expression
(30), whereas long-term GHRH antiserum treatment
has been found to decrease GHRH receptor mRNA
expression (19). Furthermore, short-term GHRH treatment downregulates GHRH receptor mRNA expression of pituitary cells cultured in serum-free medium
(1). However, animals that chronically overexpress
GHRH in vivo maintain high circulating levels of GH
and continue to secrete GH in response to exogenous
GHRH treatment (26). These apparently disparate
findings raise the possibility of a complex regulatory
system in which the capacity of GHRH to influence
expression of its pituitary receptor may depend on the
duration of GHRH exposure, the ambient cell culture
conditions, and/or the age of the animal studied. Although these factors are potentially critical in determining the effect of GHRH on its pituitary receptor,
they have not been systematically assessed.
This study focuses on defining the regulation of
GHRH receptor gene expression by GHRH in developing and mature pituitaries by means of in vitro and in
vivo approaches. The specific aims were to 1) define
whether the effect of GHRH on GHRH receptor mRNA
expression is determined by the duration of exposure to
GHRH, 2) determine whether the capacity of the pituitary to regulate GHRH receptor in response to GHRH
is developmentally regulated, 3) determine whether
the effect of GHRH on GHRH receptor mRNA expression is influenced by the presence of serum in the
culture medium, and 4) investigate the role of GHRH
in maintaining GHRH receptor mRNA expression during key stages of development.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
http://www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 11, 2017
Lasko, Catherine M., Andrew I. Korytko, William B.
Wehrenberg, and Leona Cuttler. Differential GH-releasing hormone regulation of GHRH receptor mRNA expression in the rat pituitary. Am J Physiol Endocrinol Metab
280: E626–E631, 2001.—To understand the capacity of
growth hormone-releasing hormone (GHRH) to regulate
expression of the GHRH receptor, we studied the effects of
GHRH on GHRH receptor mRNA expression in immature
and adult rats by use of pituitary cell culture and immunoneutralization approaches. Pituitary cell cultures from
neonatal (2-day-old) and adult (70-day-old) rats were
treated with GHRH for 4, 24, or 72 h. The effect of GHRH
on GHRH receptor mRNA expression depended on the
duration of GHRH exposure in both age groups; short-term
(4 h) GHRH treatment significantly reduced GHRH receptor mRNA expression (P ⬍ 0.05), whereas intermediate
treatment (24 h) restored GHRH receptor mRNA to basal
levels, and long-term treatment (72 h) stimulated GHRH
receptor mRNA expression (P ⬍ 0.02). The long-term stimulatory effect of GHRH on GHRH receptor mRNA expression required the presence of serum in the culture medium,
and, in the absence of serum, the stimulatory effect was
completely abolished. Moreover, the capacity of the pituitary to increase GHRH receptor mRNA expression in
response to 72-h GHRH treatment was age dependent,
with neonatal pituitaries exhibiting a much greater stimulatory effect than adult pituitaries (P ⬍ 0.025). Immunoneutralization of endogenous GHRH significantly reduced
GHRH receptor mRNA expression in neonatal (P ⬍ 0.004),
juvenile (P ⬍ 0.003), and mature (P ⬍ 0.004) pituitaries
compared with age-matched controls. Taken together,
these results indicate that GHRH is a potent regulator of
GHRH receptor gene expression in immature and mature
pituitaries; however, the nature and direction of GHRH
regulation of its receptor depend significantly on several
variables, including the duration of GHRH exposure, the
presence of permissive components in serum, and the developmental stage of the pituitary.
growth hormone-releasing hormone; development; neonate
DIFFERENTIAL REGULATION OF GHRH RECEPTOR BY GHRH
MATERIALS AND METHODS
In Vitro Studies
RNA by ribonuclease protection assay. GH mRNA was assessed in 2 ␮g of total RNA by Northern blot analysis, as
described previously (25). All protected or hybridized bands
were quantified by radioimaging, and data were expressed as
counts per minute per milligram of total RNA relative to that
of controls.
Statistical Analysis
A minimum of three totally independent experiments was
conducted for each treatment and age group for both in vitro
and in vivo study protocols. For each outcome measure, the
mean value for GHRH receptor mRNA expression in each
experiment was utilized in the data analysis. GHRH receptor
mRNA expression under experimental conditions was expressed as a percentage of that under vehicle-treated (control) conditions. Comparisons between groups were evaluated by paired or independent Student’s t-test, as
appropriate. A P value ⬍0.05 was equated with a significant
statistical difference.
RESULTS
In Vitro Studies
Effect of GHRH treatment duration on GHRH receptor mRNA expression in neonates. GHRH receptor
mRNA expression in neonatal pituitary cells was dependent on the duration of GHRH treatment (Fig. 1).
Short-term (4 h) exposure to GHRH reduced GHRH
receptor mRNA expression to 66 ⫾ 12% of controls (P ⬍
0.045), and 24-h exposure to GHRH restored GHRH
receptor mRNA expression to levels equivalent to those
of controls (153 ⫾ 28% of controls). However, 72-h
In Vivo Studies
Animals and experimental protocol. Rats were studied at
postnatal day 1 (neonate), day 25 (juvenile), or day 70 (adult).
In each age group, rats were treated with a highly specific
antiserum to GHRH, in a dose known to be immunoneutralizing (10 ␮l/10 g for neonates and 250 ␮l for juveniles and
adults; kindly provided by Dr. W. Wehrenberg, Clemson
University) or an equal volume of normal rabbit serum (controls) subcutaneously daily for 14 days (42). Body weights
were measured daily. After the 14-day treatment period,
animals were killed by decapitation, and the pituitaries were
removed and weighed to the nearest milligram, and total
RNA was processed as described above. GHRH receptor
mRNA expression was assessed in 20 ␮g of total pituitary
Fig. 1. Expression of the pituitary growth hormone-releasing hormone (GHRH) receptor mRNA by pituitary cell cultures of neonatal
rats is influenced by duration of exposure to GHRH. Pituitary cultures were treated for 4, 24, or 72 h with GHRH (10 nM) or vehicle
(controls) in serum-containing medium. Total RNA was analyzed for
GHRH receptor mRNA by ribonuclease protection assay. n.s., Not
significant. Data represent the means ⫾ SE of 3 separate experiments; a representative experiment is shown.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 11, 2017
Animals and primary pituitary cell culture. Neonatal
[2-day-old (day 0, day of birth)] and young adult male (70day-old) Sprague-Dawley rats (Zivic Miller, Zelienople, PA)
were studied. These ages correspond to major changes in
circulating GH levels (35, 40). All rats were maintained on a
12:12-h light (0600–1800)-dark cycle at constant ambient
temperature and were provided food (standard lab chow) and
water ad libitum. Animals were killed between 0900 and
1100 by decapitation. Whole pituitaries were removed, immediately placed in ice-cold Dulbecco’s Modified Eagle Medium (DMEM) with 25 mM HEPES and 0.3% bovine serum
albumin (BSA), and prepared for primary cell culture as
previously described (40). Cells were maintained in serumcontaining medium (DMEM supplemented with MEM nonessential amino acids, L-glutamine, 10% horse serum, 2.5%
fetal bovine serum, nystatin, and gentamicin) as described
(16), and plated in 35-mm wells at a density of 3 ⫻ 106
cells/well for 24 h before treatment.
Experimental protocol. To assess the influence of duration
of exposure to GHRH on expression of GHRH receptor in
neonatal and adult rats, pituitary cell cultures from both age
groups were treated with 10 nM GHRH (Peninsula Laboratories, Belmont, CA) or vehicle (controls) for 4, 24, or 72 h,
beginning 1 day after cell plating. The treatment dose of
GHRH (10 nM) elicits a maximal GH secretory response in
both perinatal and adult rat pituitaries (12). For the 72-h
treatment period, medium (containing 10 nM GHRH or vehicle) was replaced every 24 h. After the treatment periods,
total RNA was isolated by acid guanidinium thiocyanatephenol-chloroform extraction (11) and retained at ⫺70°C for
analysis. GHRH receptor mRNA expression was determined
in equal amounts of total RNA by ribonuclease protection
assay, as previously described (25). Protected bands were
quantified by radioimaging (AMBIS, San Diego, CA), and
data were expressed as counts per minute relative to that of
controls.
To assess the role of serum on basal and GHRH-mediated
GHRH receptor mRNA expression, pituitary cells from adult
rats were prepared and maintained in serum-containing medium for 24 h as described above. Cells were subsequently
washed and maintained in either the serum-containing medium or a defined serum-free medium [DMEM with 0.2%
BSA, 10 mM HEPES, parathyroid hormone 200 ng/l, glucagon 10 ng/l, transferrin 10 mg/l, penicillin-streptomycin, and
nystatin (24, 45, 46)] and treated with either 10 nM GHRH or
vehicle (controls) for 72 h. After the treatment period, total
RNA was extracted, and GHRH receptor mRNA expression
was assessed as described above.
E627
E628
DIFFERENTIAL REGULATION OF GHRH RECEPTOR BY GHRH
In Vivo Studies
Treatment with GHRH antiserum exerted pronounced effects on the GH axis in neonatal, juvenile,
Fig. 3. Neonatal pituitaries exhibit a marked increase in GHRH
receptor mRNA expression after 72 h of GHRH treatment in serumcontaining medium, compared with adult pituitaries. Data represent
the means ⫾ SE of 3 separate experiments as depicted in Figs. 1
and 2.
and adult rats. GHRH antiserum significantly reduced
GHRH receptor mRNA abundance in all age groups
(Fig. 5). GHRH receptor mRNA expression fell to 62 ⫾
5% of controls in neonates (P ⬍ 0.004), 59 ⫾ 8% of
controls in juveniles (P ⬍ 0.003), and 60 ⫾ 9% of
controls in adults (P ⬍ 0.004). In addition, treatment
with GHRH antiserum decreased pituitary weight in
neonatal (79 ⫾ 3% of controls, P ⬍ 0.002) and juvenile
rats (65 ⫾ 4% of controls, P ⬍ 0.001) and decreased
body weight in juvenile (59 ⫾ 3% of controls, P ⬍ 0.001)
and adult rats (43 ⫾ 7% of controls, P ⬍ 0.0013).
Pituitary GH mRNA levels were significantly reduced
by GHRH antiserum treatment in all groups relative to
controls (55 ⫾ 4% of controls in neonates, P ⬍ 0.0062;
26 ⫾ 5% of controls in juveniles, P ⬍ 0.0001; 64 ⫾ 4%
of controls in adults, P ⬍ 0.036).
DISCUSSION
Fig. 2. Expression of the pituitary GHRH receptor mRNA in adult
rats is also influenced by duration of exposure to GHRH. Pituitary
cell cultures were treated for 4, 24, or 72 h with GHRH (10 nM) or
vehicle (controls) in serum-containing medium. Total RNA was analyzed for GHRH receptor mRNA by ribonuclease protection assay.
Data represent the means ⫾ SE of 3 separate experiments.
GH secretion from pituitary somatotrophs is regulated primarily by two hypothalamic hormones, GHRH
and somatostatin. GHRH stimulates GH synthesis and
secretion after its binding to the GHRH receptor,
whereas somatostatin inhibits GH secretion via its
interaction with one or more somatostatin receptors (8,
38). In addition, the recent cloning of the GH-secretagogue receptor and identification of an endogenous
ligand strongly implicate another endogenous regulator of GH secretion (20–22, 29). Collectively, these
hormones are believed to establish normal circulating
GH levels and to direct rhythmic pulses of GH secretion (41). Central to each of their actions are their
respective pituitary receptors.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 11, 2017
GHRH treatment of neonatal pituitary cells resulted in
a dramatic increase in GHRH receptor mRNA levels
over vehicle-treated controls (363 ⫾ 65%, P ⬍ 0.015).
Effect of GHRH treatment duration on GHRH receptor mRNA expression in adults. The effect of GHRH on
GHRH receptor mRNA expression was also markedly
dependent on the duration of GHRH treatment in
adult pituitaries (Fig. 2). Short-term (4 h) treatment
with GHRH reduced expression of GHRH receptor
mRNA to 62 ⫾ 10% of controls (P ⬍ 0.02). However,
24-h GHRH treatment reestablished GHRH receptor
mRNA expression to levels similar to those of controls.
Moreover, 72-h GHRH treatment stimulated GHRH
receptor mRNA expression significantly (176 ⫾ 21% of
controls, P ⬍ 0.01). The induction of GHRH receptor
mRNA expression in neonatal pituitaries, however, far
exceeded that in adult pituitaries treated identically
(363 ⫾ 65 and 176 ⫾ 21% of controls, respectively, P ⬍
0.025; Fig. 3).
Effect of serum in the culture medium on GHRH
receptor mRNA expression. GHRH receptor mRNA expression was markedly affected by the presence of
serum in the culture medium (Fig. 4). Basal expression
of GHRH receptor mRNA by adult cells cultured in
serum-free medium was 65 ⫾ 5% of that by cells
cultured in serum-containing medium (P ⬍ 0.002).
When cells were cultured in serum-containing medium, 72-h GHRH treatment increased GHRH receptor mRNA expression (P ⬍ 0.01), but the stimulatory
effect of GHRH was completely abolished when cells
were cultured in serum-free medium (Fig. 4).
DIFFERENTIAL REGULATION OF GHRH RECEPTOR BY GHRH
This study focuses on the homologous regulation of
pituitary GHRH receptor by GHRH. Precedents show
that control of receptors by their endogenous ligands
represents a fundamental mechanism for biological
regulation and frequently has therapeutic ramifications (13, 27, 36). The current data indicate that GHRH
influences expression of its pituitary receptor in a multifaceted manner, with duration of exposure to GHRH,
ambient culture medium, and age as key determinant
variables.
Our results indicate that the duration of GHRH
exposure influences the effect of GHRH on GHRH
receptor mRNA expression. Although short-term exposure to GHRH reduces GHRH receptor mRNA expression, extended GHRH exposure (24 h) restores receptor
mRNA expression to levels comparable to those of
controls. Furthermore, more prolonged exposure to
GHRH (72 h) stimulates GHRH receptor mRNA expression. These results suggest that GHRH exerts a
short-term suppressive effect and a longer-term inductive effect on GHRH receptor mRNA levels. Earlier
studies indicate that continuous GHRH treatment in
vivo or in vitro initially stimulates GH levels but that
GH responses then decline considerably (2, 5–7, 17,
43). In previous work, we also found that, with prolonged exposure of pituitary cell cultures to GHRH, GH
concentrations in the medium rise and, in neonates,
continue above basal; however, the GH secretory response to GHRH declines (12). Although depletion of
GH stores may play a role in the reduction of GHRHstimulated GH release after prolonged exposure to
GHRH (9, 12), it has been suggested that a concomitant decrease in GHRH receptor mRNA expression or
function (4, 5, 44) contributes to the fall in GH secretory response. The current results suggest, however,
that, based on the stimulatory response of GHRH receptor mRNA expression exhibited after prolonged
GHRH exposure, a decrease in GHRH-mediated stimulation of GH secretion after prolonged exposure to
GHRH is not likely due to a direct decrease in gene
expression of GHRH receptor.
The results also indicate that the presence of serum in
the culture medium plays a permissive role in maintaining basal levels of the GHRH receptor and in mediating
the stimulatory effect of GHRH on GHRH receptor
mRNA expression. The precise serum components that
are required for GHRH to exert its stimulatory effect are
not known. Studies from our laboratory and others (24,
28, 31–33) have demonstrated that thyroid hormone and
glucocorticoids, both present in serum, can stimulate
GHRH receptor mRNA expression. We have previously
found, however, that the stimulatory effect of thyroid
hormone and glucocorticoids on the GHRH receptor is
similar in neonates and adults (23), suggesting that these
hormones are not likely responsible for the observed
differential effect of age on GHRH-induced GHRH receptor mRNA expression. It is further unlikely that standard
nutrients in serum-free medium inhibited the stimulatory effect of GHRH or that serum-free medium damaged
the cells, because the defined medium is standard for
studies of somatotroph function, and other regulatory
agents alter GHRH receptor mRNA expression in such
medium (24, 45, 46). Taken together with the previous
data, the current results suggest a combinatorial process
in the physiological regulation of GHRH receptor gene
expression.
Fig. 5. GHRH antiserum treatment (14 days) decreases GHRH receptor mRNA expression in pituitaries of neonatal, juvenile, and
adult rats. Data are expressed as percentages of the respective
age-matched controls (normal rabbit serum treatment) and represent the means ⫾ SE of ⱖ3 independent samples.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 11, 2017
Fig. 4. The ambient culture medium greatly affects basal and
GHRH-induced GHRH receptor mRNA expression. Basal GHRH
receptor mRNA expression was reduced in pituitary cells cultured
for 72 h in serum-free medium compared with cells cultured in
serum-containing medium (*P ⬍ 0.002). Whereas GHRH treatment
increased GHRH receptor mRNA expression in cells cultured in
serum-containing medium (†P ⬍ 0.01), the stimulatory effect was
blocked when cells were treated with GHRH in serum-free medium
(‡P ⫽ n.s.). GHRH receptor mRNA abundance is expressed as percentage of that in serum-containing medium alone. Data represent
the means ⫾ SE of 3 separate experiments; a representative experiment is shown.
E629
E630
DIFFERENTIAL REGULATION OF GHRH RECEPTOR BY GHRH
This work was supported by grants from the National Institutes of
Health (L. Cuttler), Eli Lilly, and the Rainbow Babies and Children’s
Hospital Board of Trustees (A. Korytko).
REFERENCES
1. Aleppo G, Moskal SF, De Grandis PA, Kineman RD, and
Frohman LA. Homologous down-regulation of growth hormonereleasing hormone receptor messenger ribonucleic acid levels.
Endocrinology 138: 1058–1065, 1997.
2. Badger TM, Millard WJ, McCormick GF, Bowers CY, and
Martin JB. The effects of growth hormone (GH)-releasing peptides on GH secretion in perifused pituitary cells of adult male
rats. Endocrinology 115: 1432–1438, 1984.
3. Barinaga M, Bilezikjian LM, Vale WW, Rosenfeld MG, and
Evans RM. Independent effects of growth hormone releasing
factor on growth hormone release and gene transcription. Nature
314: 279–281, 1985.
4. Bilezikjian LM, Seifert H, and Vale W. Desensitization to
growth hormone-releasing factor (GRF) is associated with downregulation of GRF-binding sites. Endocrinology 118: 2045–2052,
1986.
5. Bilezikjian LM and Vale WW. Chronic exposure of cultured
rat anterior pituitary cells to GRF causes partial loss of responsiveness to GRF. Endocrinology 115: 2032–2034, 1984.
6. Blumenfeld Z, Tapaneinen P, Kaplan SL, Grumbach MM,
and Jaffe RB. Partial loss of responsiveness of human fetal
pituitary cells to hGHRH after chronic exposure. Acta Endocrinol 121: 721–726, 1989.
7. Borges JL, Uskavitch DR, Kaiser DL, Cronin MJ, Evans
WS, and Thorner MO. Human pancreatic growth hormonereleasing factor-40 (hpGRF-40) allows stimulation of GH release
by TRH. Endocrinology 113: 1519–1521, 1983.
8. Bruno JF, Xu Y, and Berelowitz M. Somatostatin regulates
somatostatin receptor subtype mRNA expression in GH3 cells.
Biochem Biophys Res Commun 202: 1738–1743, 1994.
9. Ceda GP and Hoffman AR. Growth hormone-releasing factor
desensitization in rat anterior pituitary cells in vitro. Endocrinology 116: 1334–1340, 1985.
10. Cella SG, Locatelli V, de Gennaro V, Puggioni R, Pintor C,
and Muller EE. Human pancreatic growth hormone (GH)releasing hormone stimulates GH synthesis and release in infant rats. An in vivo study. Endocrinology 116: 574–577, 1985.
11. Chomczynski P and Sacchi N. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.
12. Collins BJ, Szabo M, and Cuttler L. Differential desensitization response of the neonatal and adult rat somatotroph to
growth hormone-releasing hormone and phorbol ester. Mol Cell
Endocrinol 117: 75–81, 1996.
13. Conn PM and Crowley WF Jr. Gonadotropin-releasing hormone and its analogs. Annu Rev Med 45: 391–405, 1994.
14. Cuttler L. The regulation of growth hormone secretion. Endocrinol Metab Clin North Am 25: 541–571, 1996.
15. Cuttler L, Glaum SR, Collins BA, and Miller RJ. Calcium
signalling in single growth hormone-releasing factor-responsive
pituitary cells. Endocrinology 130: 945–953, 1992.
16. Cuttler L, Welsh JB, and Szabo M. The effect of age on
somatostatin suppression of basal, growth hormone (GH)-releasing factor-stimulated, and dibutyryl adenosine 3⬘,5⬘-monophosphate-stimulated GH release from rat pituitary cells in monolayer culture. Endocrinology 119: 152–158, 1986.
17. De Zegher F, Bettendorf M, Grumbach MM, and Kaplan
SL. Hormone ontogeny in the ovine fetus. XXV. Somatotrope
desensitization to growth hormone releasing factor (GRF) independent of short-latency, ultrashortloop GH feedback. Neuroendocrinology 52: 429–433, 1990.
18. Ezzat S, Laks D, Oster J, and Melmed S. Growth hormone
regulation in primary fetal and neonatal rat pituitary cell cultures: the role of thyroid hormone. Endocrinology 128: 937–943,
1991.
19. Horikawa R, Hellmann P, Cella SG, Torsello A, Day RN,
Muller EE, and Thorner MO. Growth hormone-releasing factor (GRF) regulates expression of its own receptor. Endocrinology 137: 2642–2645, 1996.
20. Hosoda H, Kojima M, Matsuo H, and Kangawa K. Purification and characterization of rat des-Gln14-Ghrelin, a second
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 11, 2017
Several in vivo and in vitro studies (10, 12, 18, 34, 39,
40) have demonstrated that pituitaries of perinatal
animals exhibit heightened GH secretory response to
GHRH compared with adult pituitaries and that perinatal pituitaries are relatively resistant to GHRH desensitization compared with mature pituitaries. We
have previously demonstrated that expression of rat
pituitary GHRH receptor mRNA is developmentally
determined, with high levels in neonates and declining
levels later in life; this pattern of GHRH receptor
mRNA expression may contribute to classic developmental changes in circulating GH levels and to agedependent pituitary responsiveness to GHRH (25).
Therefore, it was of considerable interest to explore
whether the capacity of GHRH to modulate GHRH
receptor mRNA expression is developmentally determined. The results indicate that neonatal pituitaries
exhibit a much greater induction of GHRH receptor
mRNA expression after 72 h of GHRH treatment than
adult pituitaries. The heightened capacity of the immature pituitary to stimulate GHRH receptor gene
expression in response to GHRH may contribute to the
previously observed differential responsiveness of neonatal and adult pituitaries to GHRH.
Although in vitro models provide important information on the capacity of hormones to regulate gene
expression, a potential drawback is that they involve
an artificial environment that may not be applicable to
in vivo effects. Therefore, we also sought to determine
whether GHRH is a mediator of GHRH receptor mRNA
expression throughout development in vivo. Previous
studies (19) have shown that passive immunoneutralization of endogenous GHRH decreases GHRH receptor mRNA expression in the immature rat pituitary.
The current findings demonstrate that GHRH is necessary and permissive for maintaining normal GHRH
receptor mRNA expression throughout development.
Of particular interest was the potent effect of GHRH
antiserum on decreasing GH mRNA expression in juvenile rats. The dramatic decrease in GH mRNA, coupled with the decrease in GHRH receptor mRNA, suggests that GHRH is critically important in mediating
pituitary function during sexual maturation.
In summary, the results of this study indicate that
GHRH is a key mediator of GHRH receptor mRNA
expression both in vitro and in vivo. Moreover, the
results demonstrate that the nature and direction of
GHRH regulation of its receptor depend significantly
on several variables, including the duration of GHRH
exposure, the ambient pituitary cell environment, and
the age of the animal. Recognition of the combinatorial
process by which GHRH influences expression of its
pituitary receptor is essential to developing an understanding of the physiological regulation of the GHRH
receptor and the GH axis.
DIFFERENTIAL REGULATION OF GHRH RECEPTOR BY GHRH
21.
22.
23.
24.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
growth hormone-releasing hormone receptor messenger ribonucleic acid expression by glucocorticoids in MtT-S cells and in the
pituitary gland of fetal rats. Endocrinology 140: 2763–2770,
1999.
Ohmura E, Jansen A, Chernick V, Winter J, Friesen HG,
Rivier J, and Vale W. Human pancreatic growth hormone
releasing factor (hpGRF-1–40) stimulates GH release in the
ovine fetus. Endocrinology 114: 299–301, 1984.
Ojeda SR and Jameson HE. Developmental patterns of
plasma and pituitary growth hormone (GH) in the female rat.
Endocrinology 100: 881–889, 1977.
Pozzoli G, Bilezikjian LM, Perrin MH, Blount AL, and
Vale WW. Corticotropin-releasing factor (CRF) and glucocorticoids modulate the expression of type 1 CRF receptor messenger
ribonucleic acid in rat anterior pituitary cell cultures. Endocrinology 137: 65–71, 1996.
Saji M, Akamizu T, Sanchez M, Obici S, Avvedimento E,
Gottesman ME, and Kohn LD. Regulation of thyrotropin
receptor gene expression in rat FRTL-5 thyroid cells. Endocrinology 130: 520–533, 1992.
Schonbrunn A, Gu YZ, Dournard P, Beaudet A, Tannenbaum GS, and Brown PJ. Somatostatin receptor subtypes:
specific expression and signaling properties. Metabolism 45:
8–11, 1996.
Styne DM. The growth hormone secretory response to growth
hormone releasing factor in the developing rhesus monkey.
J Med Primatol 20: 338–344, 1991.
Szabo M and Cuttler L. Differential responsiveness of the
somatotroph to growth hormone-releasing factor during early
neonatal development in the rat. Endocrinology 118: 69–73,
1986.
Tannenbaum GS and Ling N. The interrelationship of growth
hormone (GH)-releasing factor and somatostatin in generation of
the ultradian rhythm of GH secretion. Endocrinology 115: 1952–
1957, 1984.
Wehrenberg WB, Bloch B, and Philips BJ. Antibodies to
growth hormone-releasing factor inhibit somatic growth. Endocrinology 115: 1218–1220, 1984.
Wehrenberg WB, Brazeau P, Ling N, Textor G, and Guillemin R. Pituitary growth hormone response in rats during a
24-h infusion of growth hormone-releasing factor. Endocrinology
114: 1613–1616, 1984.
Wehrenberg WB, Seifert H, Bilezikjian LM, and Vale W.
Down-regulation of growth hormone releasing factor receptors
following continuous infusion of growth hormone releasing factor
in vivo. Neuroendocrinology 43: 266–268, 1986.
Welsh JB, Cuttler L, and Szabo M. Ontogeny of the in vitro
growth hormone stimulatory effect of thyrotropin-releasing hormone in the rat. Endocrinology 119: 2368–2375, 1986.
Yamashita S and Melmed S. Insulin-like growth factor I
action on rat anterior pituitary cells: suppression of growth
hormone secretion and messenger ribonucleic acid levels. Endocrinology 118: 176–182, 1986.
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.5 on May 11, 2017
25.
endogenous ligand for the growth hormone secretagogue receptor. J Biol Chem 275: 21995–22000, 2000.
Howard AD, Feighner SD, Cully DF, Arena JP, Liberator
PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC,
Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee
KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M,
Heavens R, Rigby M, Sirinathsinghji DJS, Dean DC, Melillo DG, and Van der Ploeg LH. A receptor in pituitary and
hypothalamus that functions in growth hormone release. Science
273: 974–977, 1996.
Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, and
Kangawa K. Ghrelin is a growth-hormone-releasing acylated
peptide from stomach. Nature 402: 656–660, 1999.
Korytko AI and Cuttler L. Developmental regulation of pituitary growth hormone-releasing hormone receptor gene expression by thyroid hormone and glucocorticoids. Proc 79th Ann Mtg
Endocrine Soc Minneapolis, 1997.
Korytko AI and Cuttler L. Thyroid hormone and glucocorticoid regulation of pituitary growth hormone-releasing hormone
receptor gene expression. J Endocrinol 152: R13–R17, 1997.
Korytko AI, Zeitler P, and Cuttler L. Developmental regulation of pituitary growth hormone-releasing hormone receptor
gene expression in the rat. Endocrinology 137: 1326–1331, 1996.
Kovacs M, Kineman RD, Schally AV, Zarandi M, Groot K,
and Frohman LA. Effects of antagonists of growth hormonereleasing hormone (GHRH) on GH and insulin-like growth factor
I levels in transgenic mice overexpressing the human GHRH
gene, an animal model of acromegaly. Endocrinology 138: 4536–
4542, 1997.
Lalli E and Sassone-Corsi P. Thyroid-stimulating hormone
(TSH)-directed induction of the CREM gene in the thyroid gland
participates in the long-term desensitization of the TSH receptor. Proc Natl Acad Sci USA 92: 9633–9637, 1995.
Lam KS, Lee MF, Tam SP, and Srivastava G. Gene expression of the receptor for growth-hormone-releasing hormone is
physiologically regulated by glucocorticoids and estrogen. Neuroendocrinology 63: 475–480, 1996.
McKee KK, Palyha OC, Feighner SD, Hreniuk DL, Tan CP,
Phillips MS, Smith RG, Van der Ploeg LH, and Howard
AD. Molecular analysis of rat pituitary and hypothalamic
growth hormone secretagogue receptors. Mol Endocrinol 11:
415–423, 1997.
Miki N, Ono M, Murata Y, Ohsaki E, Tamitsu K, Yamada
M, and Demura H. Regulation of pituitary growth hormonereleasing factor (GRF) receptor gene expression by GRF. Biochem Biophys Res Commun 224: 586–590, 1996.
Miller TL and Mayo KE. Glucocorticoids regulate pituitary
growth hormone-releasing hormone receptor messenger ribonucleic acid expression. Endocrinology 138: 2458–2465, 1997.
Morpurgo B, Dean CE, and Porter TE. Identification of the
blood-borne somatotroph-differentiating factor during chicken
embryonic development. Endocrinology 138: 4530–4535, 1997.
Nogami H, Inoue K, Moriya H, Ishida A, Kobayashi S,
Hisano S, Katayama M, and Kawamura K. Regulation of
E631